The long-term goal of this project is to understand the role of membrane traffic in the cellular action of vasopressin (AVP). While it has become clear that the water channels responsible for the hydroosmotic response to AVP shuttle between a cytoplasmic vesicular location and the apical membrane in response to hormone, important features of this membrane traffic in acute and long-term regulation of water permeability remain unsolved. By exploiting the availability of cultured cortical collecting tubule cells that display the AQP-2 water channel and cAMP responses to AVP, it will be possible to determine if long-term exposure to AVP can directly enhance biogenesis of these channels as assessed by quantitative immunoassay, immunoblotting and immunolocalization. A combination of confocal and electron microscopic localization studies using newly available probes will be used to characterize the dynamics of the AQP-CD water channel's insertion, retrieval and recycling during sustained and subsequent responses. The role of membrane traffic in AVP responses will also be evaluated by electron microscopic localization of antibodies to AQP channels and clathrin in cultured cells. Label-fracture methods will be used to localize antibodies with respect to intramembrane structures and clathrin to determine if water channels are restricted to cluster domains or if they can occur separate from cluster-clathrin domains. Similar strategies applied to the toad urinary bladder will allow a localization of candidate water channel components with respect to IMP aggregates and retrieved components. The possible role of vesicular traffic in the regulation of Na+ transport will be examined using newly available antibodies to the epithelial Na+ channel (rENaC) and the Na+,K+,Cl- cotransporter. The transporters will be immunolocalized at both the light and electron microscopic level. These studies will determine if a pool of intracellular vesicles is available in responsive cells to participate in hormonal responses and whether the pool of such vesicles is enhanced by conditioning through AVP exposure.